Aspects of the mechanism of action of local anesthetics on the sarcoplasmic reticulum of skeletal muscle.

1. The effect was studied of local anesthetics (tetracaine, dibucaine, procaine and xylocaine) on the forward and the backward reactions of the calcium pump of skeletal muscle sarcoplasmic reticulum. 2. The inhibition of the rate of calcium uptake, the rate of calcium-dependent ATP splitting and the rate of calcium-dependent ATP-ADP phosphate exchange by sarcoplasmic reticulum in the presence of the above drugs is at least partially due to the inhibition of the phosphoprotein formation from ATP. 3. The rate of the ADP-induced calcium release from sarcoplasmic reticulum and the rate of ATP synthesis driven by the calcium efflux are inhibited on account of a reduction of the phosphoprotein formation by orthophosphate. 4. The phosphorylation of calcium transport ATPase by either ATP or orthophosphate is diminished by the local anesthetics owing to a reduction in the apparent calcium affinity of sarcoplasmic reticulum emmbranes on the outside and on the inside, respectively. 5. The drug-induced calcium efflux from calcium-preloaded sarcoplasmic reticulum vesicles, a reaction not requiring ADP, is probably not mediated by calcium transport ATPase.     

Calcium releasing action of quercetin on sarcoplasmic reticulum from frog skeletal muscle

The release of Ca by quercetin from the sarcoplasmic reticulum has been claimed to be a result of the well-known inhibition of Ca2+-ATPase activity, or to be due to an intrinsic property of quercetin. To get a clearer understanding of the effect of quercetin, we examined it using fragmented sarcoplasmic reticulum (FSR) from bullfrog skeletal muscle. The rapid phase of Ca release (hereafter simply referred to as "Ca release") from loaded FSR was almost completed within 5 s after addition of quercetin in the presence of ATP. It cannot be ascribed to the inhibition of Ca2+-ATPase activity on the basis of following findings. First, when Ca uptake was driven by carbamylphosphate, no or little Ca release was observed in marked contrast to a stronger reduction in the rate of Ca uptake. Secondly, procaine reverses the Ca releasing action of quercetin, whereas it show a synergistic action in the inhibition of Ca2+-ATPase activity. Thirdly, HFSR released more Ca than LFSR, while the Ca2+-ATPase activities of both fractions were inhibited to a similar extent. The Ca release by quercetin is enhanced by ATP or beta, gamma-methylene adenosine triphosphate, and decreased by procaine or a high concentration of Mg2+. In the presence of 2.5 mM caffeine, the amount of Ca2+ released by quercetin was decreased, and the dose-effect relationship was shifted to higher doses of quercetin. This indicates that quercetin and caffeine probably overlap in the site(s) of the action, but that quercetin is dissimilar from halothane in the mode of its Ca-releasing action.     

Microarray profiling of skeletal muscle sarcoplasmic reticulum proteins

Microarrays were developed to profile the level of proteins associated with calcium regulation in sarcoplasmic reticulum (SR) isolated from porcine Longissimus muscle. The microarrays consisted of SR preparations printed onto to glass slides and probed with monoclonal antibodies to 7 target proteins. Proteins investigated included: ryanodine receptor, (RyR), dihydropyridine receptor, (DHPR), triadin (TRI), calsequestrin (CSQ), 90 kDa junctional protein (JSR90), and fast-twitch and slow-twitch SR calcium ATPases (SERCA1 and SERCA2). Signal from a fluorescently-labeled detection antibody was measured and quantitated using a slide reader. The microarray developed was also employed to profile Longissimus muscle SR proteins from halothane genotyped animals. Significant (P<0.05) reductions in levels of several proteins were found including: RyR, CSQ, TRI, DHPR and SERCA2 in SR samples from halothane positive animals. The results illustrate the potential of microarrays as a tool for profiling SR proteins and aiding investigations of calcium regulation.     

Spontaneous calcium release from sarcoplasmic reticulum. Effect of local anesthetics

Spontaneous calcium release from purified light sarcoplasmic reticulum has been previously described (Palade, P., Mitchell, R. D., and Fleischer, S. (1983) J. Biol. Chem. 258, 8098-8107) and found to be distinct from several other forms of Ca2+ release. Ca2+ release occurs after a lag period following active Ca2+ preloading and depletion of extravesicular Ca2+. In the present study, we find that local anesthetics inhibit spontaneous Ca2+ release, in a time-dependent manner, varying considerably in the preincubation time required to exert maximal effect. At pH 7.0, hydrophilic and mostly charged local anesthetics, such as procaine, procainamide, and N-(2,6-dimethylphenyl carbamoyl methyl)triethyl ammonium bromide, inhibit Ca2+ release only after long preincubations (hours), whereas more hydrophobic local anesthetics are effective after only a short incubation (minutes) with sarcoplasmic reticulum. The more hydrophobic anesthetics take somewhat longer to reach equilibrium, as studied by inhibition of unidirectional Ca2+ efflux, and there is a direct relationship between hydrophobic partition coefficient and half-time to reach equilibrium. Agents known to inhibit permeability pathways for monovalent cations i.e. K+ channel blockers (decamethonium and n-dodecane-1, 12-N,N,N,N',N',N'-hexamethyl-bis-ammonium) or the anion blocker (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid), do not inhibit spontaneous Ca2+ release. Carbonyl cyanide m-fluorophenylhydrazone, a protonophore, and gramicidin D, a monovalent cation ionophore, have no effect on Ca2+ release whether local anesthetics are present or not, while the Ca2+ ionophore A23187 relieves inhibition of Ca2+ release by local anesthetics. Ruthenium red does not inhibit spontaneous Ca2+ release. These findings suggest that the binding site(s) for local anesthetics is located on the inner face of the sarcoplasmic reticulum membrane and that local anesthetics interact directly with a Ca2+ channel rather than with other permeability pathways which might indirectly influence Ca2+ channel gating.     

The interaction of local anesthetics with the ryanodine receptor of the sarcoplasmic reticulum

The effects of various local anesthetics (LAs) on the skeletal muscle ryanodine receptor were tested. The LAs were divided into three categories according to their effects on the binding of ryanodine to the junctional sarcoplasmic reticulum membranes. Ryanodine binding was assayed in the presence of 0.2 m NaCl and 10 m CaCl₂. Tetracaine and dibucaine inhibit the binding with half-maximal inhibition (CI₅₀) of 0.12 and 0.25 mm, respectively, while inhibition by benzocaine and procaine occurs with CI₅₀ of about 10-fold higher. Lidocaine, its analogue QX-314, and prilocaine, on the other hand, stimulate the binding up to fourfold with half-maximal stimulation occurring with about 2 mm of the drugs. Lidocaine increases both the receptor affinity for ryanodine by about fivefold and the rate of ryanodine association with its binding site by about 10-fold.Tetracaine interacts with the ryanodine receptor in a non-competitive fashion with respect to ryanodine but it competes with lidocaine for its binding site, suggesting the existence of a single site for the inhibitory and stimulatory LA.     

Calsequestrin targeting to sarcoplasmic reticulum of skeletal muscle fibers

Calsequestrin (CS) is the low-affinity, high-capacity calcium binding protein segregated to the lumen of terminal cisternae (TC) of the sarcoplasmic reticulum (SR). The physiological role of CS in controlling calcium release from the SR depends on both its intrinsic properties and its localization. The mechanisms of CS targeting were investigated in skeletal muscle fibers and C₂C₁₂ myotubes, a model of SR differentiation, with four deletion mutants of epitope (hemagglutinin, HA)-tagged CS: CS-HA24_NH2, CS-HA2D, CS-HA3D, and CS-HAHT, a double mutant of the NH₂ terminus and domain III. As judged by immunofluorescence of transfected skeletal muscle fibers, only the double CS-HA mutant showed a homogeneous distribution at the sarcomeric I band, i.e., it did not segregate to TC. As shown by subfractionation of microsomes derived from transfected skeletal muscles, CS-HAHT was largely associated to longitudinal SR whereas CS-HA was concentrated in TC. In C₂C₁₂ myotubes, as judged by immunofluorescence, not only CS-HAHT but also CS-HA3D and CS-HA2D were not sorted to developing SR. Condensation competence, a property referable to CS oligomerization, was monitored for the several CS-HA mutants in C₂C₁₂ myoblasts, and only CS-HA3D was found able to condense. Together, the results indicate that 1) there are at least two targeting sequences at the NH₂ terminus and domain III of CS, 2) SR-specific target and structural information is contained in these sequences, 3) heterologous interactions with junctional SR proteins are relevant for segregation, 4) homologous CS-CS interactions are involved in the overall targeting process, and 5) different targeting mechanisms prevail depending on the stage of SR differentiation. protein-protein interactions; oligomerization; intracellular sorting     

Calcium-permeable channel in sarcoplasmic reticulum of rabbit skeletal muscle

Membrane vesicles from sarcoplasmic reticulum of rabbit skeletal muscle were incorporated into a bilayer lipid membrane. With this system, single current fluctuation was observed in the presence of 50 mM Ba-gluconate. This channel activity was observed only in vesicles from terminal cisternae. The single channel conductance was 14.1 pS, and the channel state was almost wholly open. The open-close transition of the channel obeyed simple two-state kinetics and was voltage-independent. The ionic selectivity was also studied, and the channel showed no selectivity among Ba, Ca, Mn, and Mg. On the other hand, it was less permeable to Cs than to Ba. Based on these results, the relation of the Ca channel to excitation-contraction coupling is discussed.     

o-Phthalaldehyde activates the Ca(2+) release mechanism from skeletal muscle sarcoplasmic reticulum.

o-Phthalaldehyde (OPA) is a bifunctional reagent that forms an isoindole derivative by reacting with cysteine and lysine residues separated by approximately 0.3 nm. OPA inhibits sarcoplasmic reticulum (SR) Ca(2+)-ATPase activity at low micromolar concentrations and induces Ca(2+) release from actively loaded SR vesicles by activating the ryanodine receptor from fast twitch skeletal muscle. Both ryanodine binding and single-channel activity show a biphasic concentration dependence. At low OPA concentrations (<100 microM), ryanodine binding and single channel activity are stimulated, while at higher concentrations, a time-dependent sequential activation and inhibition of receptor binding is observed. Activation is characterized by a Ca(2+)-independent increase in maximal receptor occupancy. Data are presented to support a model in which Ca(2+) channel and ryanodine binding activity are enhanced due to an intramolecular cross-linking of nearby lysine and nonhyperreactive cysteine residues. OPA complexation with endogenous lysine residue(s) is critical for receptor activation.     

Effect of thyroxine on the function of rabbit skeletal muscle sarcoplasmic reticulum

Thyrotoxicosis in rabbits was induced by prolonged intraperitoneal injection of L-thyroxin. The development of thyroxicosis was assoiated with a decreased Ca2+ accumulation rate by sarcoplasmic reticulum (SR) fragments and a lowered Ca2+ dependent ATPase activity. As compared to the analogous parameters in normal animals. Ca2+ accumulation rate and ATPase activity of thyrotoxicosis animals decreased by 60 and 25%, respectively. The changes in the specific parameters of SR were also observed during incubation of normal SR samples in the medium containing thyroxin (10-5 M). The changes seen in SR functioning in thyrotoxicosis animals are likely to be related to structural rearrangements of lipoprotein surroundings of Ca-ATPase.     

Ca-ATPase self-fluorescence in the sarcoplasmic reticulum of the rabbit skeletal muscle

The quenching of the intrinsic protein fluorescence of sarcoplasmic reticulum Ca-ATPase from the rabbit skeletal muscles by hydrophylic (NaI, CsCl) or hydrophobic (pyrene, fluorescamine) substances has been studied. CsCl (up to 1 M) has been shown not to affect the intrinsic protein fluorescence while NaI (250 mM) quenches it at 15%, pyrene (8 mkM) decreases the intrinsic fluorescence of Ca-ATPase at 35% and fluorescamine (up to 40 mkM)--at 80%. Possible mechanisms of the interaction of the quenchers with the intrinsic fluorescence of sarcoplasmic reticulum Ca-ATPase are being discussed.     

Effects of Mojave toxin on rat skeletal muscle sarcoplasmic reticulum.

Effects of the lethal fraction (MD-9) from the venom of the Mojave rattlesnake, Crotalus scutulatus, on sarcoplasmic reticulum were investigated. The calcium sequestering activity of the vesicles was reduced by the lethal fraction and subsequent release of calcium was enhanced. These effects were observed to be dependent upon MD-9 concentration and the length of preincubation time with the vesicles. An enhanced ATPase activity that was affected by concentration and MD-9 preincubation time was also observed. Both calcium uptake and ATPase activity effects may be due to a phospholipase activity associated with the fraction.     

Effects of palmitoyl coenzyme A on rat skeletal muscle sarcoplasmic reticulum

Palmitoyl coenzyme A (PCoA) inhibits Ca2+ uptake and stimulates Ca2+-activated ATPase in sarcoplasmic reticulum vesicles. The inhibitory effect on Ca2+-uptake is referable to a stimulation of Ca2+ release which is directly correlated to the concentration of PCoA added. The comparison of the Ca2+-releasing effect of PCoA in different experimental conditions indicates that concentrations of PCoA higher than 10 microM may be disruptive for the vesicles while concentrations of PCoA lower than this value can activate a Ca2+-releasing channel or more generally can increase the membrane permeability for Ca2+.